CN109456908A - A kind of genetic engineering bacterium producing D-pantoyl lactone hydrolase and its construction method and application - Google Patents
A kind of genetic engineering bacterium producing D-pantoyl lactone hydrolase and its construction method and application Download PDFInfo
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Abstract
The invention discloses a kind of genetic engineering bacterium for producing D-pantoyl lactone hydrolase, the construction methods of the engineering bacteria are as follows: the encoding gene of D-pantoyl lactone hydrolase is transferred in host's Kluyveromyces lactis cell, the engineering bacteria is made；Nucleotide fragments containing recombinant vector pZL505-DL in the engineering bacteria, the DL sequence is as shown in SEQ ID NO:1.The present invention solves the problems, such as that D-pantoyl lactone hydrolase expression quantity existing in the prior art is low.
Description
Technical field
The present invention relates to gene engineering technology fields, more particularly, to a kind of gene work for producing D-pantoyl lactone hydrolase
The construction method of journey bacterium and its application.
Background technique
With deepening continuously for Molecular Biology research and constantly bringing forth new ideas for molecular biology method, genetic engineering skill
Art is rapidly developed, and the achievement to attract people's attention is achieved.As the important content of technique for gene engineering, gene expression technique seeps
The every field for having arrived industry, agricultural and life science thoroughly, attracts widespread attention.According to exogenous gene expression host
Difference, currently, the gene expression system developed has escherichia expression system, mammalian expression systems, Yeast expression
System etc..Wherein yeast expression system have many advantages, such as it is easy to operate, be easy to cultivate, the speed of growth it is fast, at low cost, also have big
The features such as not available for enterobacteria expression system to the posttranslational modification of foreign protein, therefore obtain more and more extensive answer
With.
Kluyveromyces lactis genetic background is clear and the technology maturation in terms of genetic manipulation, is authenticated by U.S. FDA
It is food safety level microbe, excellent protein expression system is answered extensively in food enzyme and pharmaceutical protein production
With.
D-pantoyl lactone hydrolase is the specific enzyme of chemical-enzymatic production D-VB5 calcium, is played in the production of calcium pantothenate
Important function.Leading position is occupied currently with chemical resolution method production calcium pantothenate, but chemical method is in the production point of calcium pantothenate
From the toxicity problem for generation environment pollution problem and chiral resolving agent in the process, compared with chemical method, chemical-enzymatic more meets
Environmental protection requirement and cost is relatively low.In chemical-enzymatic, the original bacteria fermentation yield for producing D-pantoyl lactone hydrolase is lower, example
The production level that such as Tang Yixin filters out the bacterial strain of one plant of production D-pantoyl lactone hydrolase is 4.65U/L (Tang Yixin, Sun Zhi
It is great, Hua Lei, wait .D- pantoic acid lactone hydrolase producing strains screening and condition of enzyme production research [J] microorganism journal, 2002,42
(1): 81-87.), in the production level of the bacterial strain Fusarium sp.BU-011 of bright and sharp equal screenings be 410U/L fermentation thalli (Yu
M R,Tan T W.Optical resolution of racemic pantolactone by Fusarium sp.BU-011
with high lactonohydrolase activity[J].Process Biochemistry,2005,40(8):2609-
2614.), therefore the strong bacterial strain of fermentation advantage is selected to carry out heterogenous expression to D-pantoyl lactone hydrolase gene to improve enzyme activity
Expression quantity, industrial application value with higher.
Summary of the invention
In view of the above-mentioned problems existing in the prior art, the applicant provides a kind of production D-pantoyl lactone hydrolases
Genetic engineering bacterium and its construction method and application.The present invention solves D-pantoyl lactone hydrolase existing in the prior art
The low problem of expression quantity.
Technical scheme is as follows:
A kind of genetic engineering bacterium producing D-pantoyl lactone hydrolase, the construction method of the engineering bacteria are as follows: by the general solution of D-
The encoding gene of acid lactone hydrolase is transferred in host's Kluyveromyces lactis cell, and the engineering bacteria is made；The engineering bacteria
The interior nucleotide fragments containing recombinant vector pZL505-DL, the DL sequence is as shown in SEQ ID NO:1.
The host is Kluyveromyces lactis GG799.
A kind of construction method for the genetic engineering bacterium producing D-pantoyl lactone hydrolase, the construction method include following step
It is rapid:
It (1) is upstream primer, SEQ ID using sequence shown in SEQ ID NO:2 using the DL sequence of synthesis as template
Sequence shown in NO:3 is that downstream primer carries PCR product and pZL505 by reaction acquisition product, then with Xho I and Kpn I
Connection obtains recombinant vector pZL505-DL after body carries out double digestion；
(2) recombinant expression carrier is expanded using competent escherichia coli cell and collect amplified production, by the expansion
Conversion host cell obtains genetic engineering bacterium after volume increase object carries out digestion with Sac II.
A kind of application for the genetic engineering bacterium producing D-pantoyl lactone hydrolase, for hydrolyzing D-pantoyl lactone or fractionation
DL- pantoic acid lactone.
The hydrolysis D-pantoyl lactone or the method for splitting DL- pantoic acid lactone are as follows: genetic engineering bacterium is seeded to training
It supports and is cultivated in base, use resin adsorption after obtaining fermentation crude enzyme liquid, enzyme preparation is made；Enzyme preparation is used to hydrolyze the general solution of D- again later
Acid lactone lactone splits DL- pantoic acid lactone.
The group of the culture medium becomes sucrose 40g/L, peptone 20g/L, beef extract 15g/L, NaCl 6g/L, KCl 6g/
L、CaCl2 3g/L、MgSO4 3g/L。
The enzyme preparation the preparation method comprises the following steps: using resin D380 as carrier, using glutaraldehyde as crosslinking agent, in Pantothenic acid
Additive amount 10~50U/g resin of ester hydrolase, adsorb pH 6.5~7.5,20~35 DEG C of adsorption temp, 3~8h of adsorption time；
Glutaraldehyde final concentration 0.1~1%, 0.5~3h of crosslinking time.
The condition of culture are as follows: under the conditions of 25~35 DEG C of temperature, 150~200rpm, incubation time is 70~90h.
The present invention is beneficial to be had the technical effect that
Genetic engineering bacterium provided by the present invention has the ability of high efficient expression D-pantoyl lactone hydrolase, expressed
D-pantoyl lactone hydrolase has the ability of effectively hydrolyzing D-pantoyl lactone or selective hydrolysis DL- pantoic acid lactone.
Genetic engineering bacterium of the present invention has higher D-pantoyl lactone hydrolase expression quantity, and final enzyme activity can reach 3~5U/
mL。
Detailed description of the invention
Fig. 1 is the D-pantoyl lactone enzyme gene that embodiment 1 optimizes；
In figure: swimming lane M, molecular weight Marker；The D-pantoyl lactone enzyme gene PCR amplification of 1,2 sequence optimisation of swimming lane produces
Object；
Fig. 2 is embodiment 1D- pantoic acid lactone enzyme clone recombinant plasmid digestion verification；
In figure: swimming lane M, molecular weight Marker；The verifying of I/Kpn of swimming lane 1,2Xho, I double digestion；
Fig. 3 is embodiment 1D- pantoic acid lactone enzyme recombinant plasmid pZL505-DL digestion verification；
In figure: swimming lane M, molecular weight Marker；Swimming lane 1, the verifying of I/Kpn of Xho, I double digestion；
Fig. 4 is 1 recombinant bacterium expression product SDS-PAGE of embodiment analysis；
In figure: M: standard protein Marker；1: recombinant bacterium fermentation supernatant；2: control strain fermented liquid supernatant；
Fig. 5 is reaction solution liquid chromatogram before and after 3 enzyme hydrolysis of embodiment；
In figure: (a) pantoic acid sample；(b) D-pantoyl lactone standard sample；(c) immobilised enzymes of D-pantoyl lactone
Reaction solution；
Fig. 6 is that 3 immobilised enzymes packed bed reactor of embodiment is catalyzed schematic diagram；
Fig. 7 is the operational stability of 3 immobilised enzymes of embodiment；
Specific embodiment
With reference to the accompanying drawings and examples, the present invention is specifically described.
PMD19-T-Simple Vector is purchased from Takara；Bacterial strain JM109 is purchased from Invitrogen；Kluyveromyces Lactis ties up ferment
Female GG799 is purchased from New England Biolabs.
In the following examples, the experimental methods for specific conditions are not specified, substantially all in accordance with common clone's handbook or institute, manufacturer
It is recommended that condition carry out experimental implementation；Manufacturer person is not specified in agents useful for same or instrument, and being can be by the normal of commercially available acquisition
Advise product.
Unless separately limiting herein, whole term used herein is logical with ordinary person of the art
The identical meanings often understood.
Embodiment 1:
A kind of genetic engineering bacterium producing D-pantoyl lactone hydrolase, construction method include the following steps:
(1) building of cloning vector pMD19-T-DL
The cDNA sequence (GenBank:AY728018.1) that D-pantoyl lactone hydrolase is found on NCBI, sends to Suzhou
Jin Weizhi biotech firm optimizes synthesis according to the codon that yeast is had a preference for, and obtains the D-pantoyl lactone hydrolase of optimization
Gene (DL), DL sequence is as shown in SEQ ID NO:1.With the gene (DL) of synthesis for template, PCR expansion is carried out with the primer of design
Increase, as a result as shown in Figure 1.Amplified production is connected with pMD19-T-Simple Vector, and building cloning vector pMD19-T-DL is simultaneously
Transformed E .coli JM109 competence, screens positive transformant using ammonia benzyl resistant panel, extracts recombinant plasmid using alkali cracking method,
Specific method carries out digestion verification, as a result as shown in Figure 2 referring to vast Tyke plasmid extraction kit.It send to Suzhou Jin Weizhi
Biotech firm be sequenced and the correct pMD19-T-DL carrier of preservation gene order.
PCR amplification system: ddH2O 45μL；1 μ L of template；2×Pfu PCR Master Mix 50μL；1 μ L of primer P1,
P2 1μL
PCR amplification condition: 95 DEG C of 5min of initial denaturation, 94 DEG C of 30s of denaturation, 55 DEG C of 30s of annealing, extend 72 DEG C of 1.5min, follow
Ring number 30 extends 72 DEG C of 10min eventually.
Primer P1 underscore part is the recognition site of Xho I, and primer P2 underscore part is the recognition site of Kpn I.
P1:5 ' GTCCTCGAGATGGCTAAACTTCCTTCTAC3’
P2:5 ' CCGGGTACCCTAATCATAAAGTTTAGGACCCGAAGCCCC 3’
Fig. 1 be optimization D-pantoyl lactone enzyme gene, the target gene fragment size cloned as seen from the figure with
It is expected that consistent.
Fig. 2 is D-pantoyl lactone enzyme clone recombinant plasmid digestion verification, and target gene is successfully connected as seen from the figure
Onto cloning vector.
(2) building of recombinant plasmid pZL505-DL
Cloning vector pMD19-T-DL and expression vector pZL505 is subjected to double digestion with Xho I, Kpn I, glue recycles DL base
Cause and pZL505 carrier are measured its nucleic acid concentration with nucleic acid quantification instrument, under the two concentration the same terms, by DL gene
It is added with pZL505 carrier according to volume ratio 1:2, T4 DNA ligase additive amount is 0.5 μ L, and total system is 10 μ L, is put into
In 0.5mL EP pipe, 16 DEG C of connection about 12h in constant-temperature metal bath are placed, then Transformed E .coli JM109 competence, selects sun
Property transformant access LB liquid medium culture about 20h, upgrading grain carry out double digestion verifying, as a result as shown in Figure 3.It can by Fig. 3
To find out target gene and expression vector successful connection.
(3) building of recombination lactic acid kluyveromyces
Recombinant plasmid pZL505-DL is linearized with Sac II, linearisation product electricity is turned into Kluyveromyces lactis competence,
And be coated with and be grown in 30 DEG C of 3~4d of culture on the YCB solid medium containing 5mM acetamide, from the region oese of about 2mm
Picking positive transformant and being inoculated in YPD fluid nutrient medium is cultivated 96 hours, carries out SDS-PAGE electrophoresis point to fermented liquid supernatant
It analyses and to measure enzyme activity result as shown in Figure 4.As seen from Figure 4, target gene is succeeded expression in host.
Enzyme activity reaction system and measuring method: by 1mL 4%D- pantoic acid lactone aqueous solution, 0.5mL pH 7.5
Tris-HCl buffer (2mol/L) and 0.5mL enzyme solution are put into 5mL EP pipe, then 30 DEG C of reaction 30min boil 15min and go out
It is living.Enzyme activity is calculated with the reduction amount of HPLC analysis D-pantoyl lactone.Added enzyme solution is changed to go out after boiling by blank control
Enzyme solution living.
Enzyme activity definition: under certain condition, enzyme amount required for 1 μm of ol D- (-) pantoic acid lactone is hydrolyzed per minute and is defined
For 1 enzyme activity unit (U).
The HPLC determination condition (United States Patent, 5275949) of D-pantoyl lactone: chromatographic column is
4.6 × 250mm × 3.5 μm ZORBAX SB-C18, flow velocity 0.5mL/min, ultraviolet detection wavelength are 215nm, 25 DEG C of column temperature, are flowed
Dynamic Phase Proportion is acetonitrile: KH2PO4(0.02mol/L)=1:19, with dilute hydrochloric acid tune pH to 3.0,10 μ L of sample volume.With this condition
The retention time of D-pantoyl lactone about 18min, the retention time of Pantothenic acid about 9.5min.As a result as shown in figure 5, by Fig. 5
As can be seen that enzymic hydrolysates are consistent with standardized products retention time, which has the energy of hydrolysis D-pantoyl lactone hydrolase
Power.
YCB solid medium: YCB powder 11.7g/L, 2mL 1.5mol/L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer
(pH 7), agar powder 20g/L, 115 DEG C of high pressure steam sterilization 20min wait addition acetamide when being cooled to 40~45 DEG C dense to end
Degree is 5mmol/L.
Embodiment 2: the producing enzyme culture of genetic engineering bacterium
The preparation of seed liquor: the 200 μ L of bacterium solution for hiding of going bail for is in YPD fluid nutrient medium, and 30 DEG C, 200r/min cultivates 36h
It is used as first order seed afterwards.In the 250mL conical flask containing 50mL YPD fluid nutrient medium, by the switching amount of 4% (volume ratio)
Be inoculated with first order seed, 30 DEG C, 200r/min culture 36h as secondary seed.
Shaking flask producing enzyme culture: medium component be sucrose 40g/L, peptone 20g/L, beef extract 15g/L, NaCl 6g/L,
KCl 6g/L、CaCl2 3g/L、MgSO43g/L, 30 DEG C, 200r/min fermentation time 85h
The measurement of biomass: indicating thalli growth amount with the dry weight of certain time unit volume (L) fermentation liquid institute mycetome,
Fermentation liquid is obtained into thallus from 5min in 10000r/min, and is washed 2 times with aseptic deionized water, 105 DEG C drying to constant weight.
It is the highest enzyme activity expression reported at present by shaking flask producing enzyme result (table 1) it is found that shaking flask culture enzyme activity is up to 5U/mL
It is horizontal.
Table 1
Embodiment 3: the application of genetic engineering bacterium
(1) preparation of immobilised enzymes
Appropriate pretreated resin D380 is put into 50mL conical flask, it is 12~80U's that every gram of resin, which is put into total enzyme activity,
Crude enzyme liquid is subsequently placed in shaking table, and 20~35 DEG C of 100~200r/min adsorb 2~6h, is pre-chilled at 4 DEG C, is added glutaraldehyde extremely
Final concentration 0.1%~1% is crosslinked 0.5~3h, finally removes resolvase with deionized water repeated flushing.Or it will pre-process in right amount
Resin D380 be put into packed bed reactor as shown in fig. 6, being inhaled with crude enzyme liquid (every gram resin total enzyme activity be 12~80U) circulation
Attached, 2~6h is pre-chilled at 4 DEG C, then is crosslinked 0.5~3h with 0.1%~1% glutaraldehyde circulation, is finally rushed repeatedly with deionized water
Resolvase is removed in washout.
(2) hydrolysis of the immobilised enzymes to DL- pantoic acid lactone
It is 20%~30% to be hydrolyzed to DL- pantoic acid lactone concentration using immobilised enzymes, and with ammonium hydroxide or hydroxide
Sodium solution adjusts pH7.0~7.5, reacts 4~6h, and immobilised enzymes is not significantly decreased using enzymatic conversion rate after 15 times, as a result
As shown in Figure 7.As seen from Figure 7, which has preferable operational stability.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of genetic engineering bacterium for producing D-pantoyl lactone hydrolase and its construction method and application
<130> 1
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<170> PatentIn version 3.3
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<213> Synotype
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gaggagtctc ttgttgagaa gccttttcat gtctacgatg aagagtttta cgacgtcatc 180
ggaaaggacc cttctttgac ccttatcgca acatcggaca ccgacccaat cttccatgag 240
gctgtcgtat ggtatcctcc tactgaagag gtcttctttg tccagaatgc tggcgctcct 300
gctgctggca ctggcttgaa caagtcttcc atcattcaga agatttccct taaggaggcc 360
gacgaggtcc gtaagggcaa gaaggatgag gtcaaggtcg cggttgttga ctcaaaccct 420
caggtcatca accctaatgg tggcacttac tacaagggca acatcatctt tgctggtgag 480
ggccaaggcg acgatgttcc ttccgccctt taccttatga accctcttcc tccttacaac 540
accaccaccc ttcttaacaa ctactttggt cgtcagttca actcccttaa cgacgtcggt 600
atcaacccta ggaacggtga cttgtacttc accgataccc tttatggata ccttcaggac 660
ttccgtcctg ttcctggtct tcgaaaccaa gtctatcgtt acaactttga caccggcgcc 720
gtcactgtcg tcgctgatga ctttaccctt cctaacggta ttggctttgg ccctgacggc 780
aagaaggtct atgtcaccga cactggtatc gctcttggct tttacggccg taacctttct 840
tcacctgcct ctgtttactc cttcgatgta aaccaggacg gtactcttca gaaccgtaag 900
acctttgctt acgtcgcgtc tttcatccct gatggtgttc ataccgactc caagggccgt 960
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Claims (8)
1. a kind of genetic engineering bacterium for producing D-pantoyl lactone hydrolase, which is characterized in that the construction method of the engineering bacteria are as follows:
The encoding gene of D-pantoyl lactone hydrolase is transferred in host's Kluyveromyces lactis cell, the engineering bacteria is made；Institute
The nucleotide fragments containing recombinant vector pZL505-DL in engineering bacteria are stated, the DL sequence is as shown in SEQ ID NO:1.
2. engineering bacteria according to claim 1, which is characterized in that the host is Kluyveromyces lactis GG799.
3. producing the construction method of the genetic engineering bacterium of D-pantoyl lactone hydrolase described in a kind of claim 1, which is characterized in that
The construction method includes the following steps:
It (1) is upstream primer, SEQ ID NO:3 institute using sequence shown in SEQ ID NO:2 using the DL sequence of synthesis as template
The sequence shown is that downstream primer by reaction obtains product, then is carried out pair with Xho I and Kpn I to PCR product and pZL505 carrier
Connection obtains recombinant vector pZL505-DL after digestion；
(2) recombinant expression carrier being expanded using competent escherichia coli cell and collecting amplified production, the amplification is produced
Conversion host cell obtains genetic engineering bacterium after object carries out digestion with Sac II.
4. producing the application of the genetic engineering bacterium of D-pantoyl lactone hydrolase described in a kind of claim 1, which is characterized in that be used for
It hydrolyzes D-pantoyl lactone or splits DL- pantoic acid lactone.
5. application according to claim 4, which is characterized in that the hydrolysis D-pantoyl lactone splits DL- pantoic acid
The method of lactone are as follows: genetic engineering bacterium is seeded in culture medium and is cultivated, resin adsorption is used after obtaining fermentation crude enzyme liquid, enzyme is made
Preparation；Enzyme preparation is used to hydrolyze D-pantoyl lactone lactone again later or splits DL- pantoic acid lactone.
6. application according to claim 5, which is characterized in that the group of the culture medium becomes sucrose 40g/L, peptone
20g/L, beef extract 15g/L, NaCl 6g/L, KCl 6g/L, CaCl2 3g/L、MgSO43g/L。
7. application according to claim 5, which is characterized in that the enzyme preparation the preparation method comprises the following steps: being with resin D380
Carrier, using glutaraldehyde as crosslinking agent, additive amount 10~50U/g resin of D-pantoyl lactone hydrolase, absorption pH 6.5~
7.5,20~35 DEG C of adsorption temp, 3~8h of adsorption time；Glutaraldehyde final concentration 0.1~1%, 0.5~3h of crosslinking time.
8. application according to claim 5, which is characterized in that the condition of culture are as follows: under the conditions of 25~35 DEG C of temperature,
150~200rpm, incubation time are 70~90h.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110452861A (en) * | 2019-07-10 | 2019-11-15 | 杭州师范大学 | A kind of genetic recombination engineering bacteria and its catalyzing and synthesizing the application in D- pantoyl internal ester |
| CN112175839A (en) * | 2020-09-30 | 2021-01-05 | 遵义医科大学 | D-pantoic acid lactone hydrolase, producing strain, genetic engineering strain and application thereof |
| CN112553226A (en) * | 2020-12-29 | 2021-03-26 | 江南大学 | Pichia pastoris gene engineering bacterium and method for preparing D-pantolactone hydrolase by using same |
| CN113046337A (en) * | 2021-03-18 | 2021-06-29 | 赤峰制药股份有限公司 | Pantolactone hydrolase mutant strain and application thereof |
| CN114457128A (en) * | 2020-09-29 | 2022-05-10 | 安徽华恒生物科技股份有限公司 | Recombinant engineering bacterium and construction method and application thereof |
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| CN110452861A (en) * | 2019-07-10 | 2019-11-15 | 杭州师范大学 | A kind of genetic recombination engineering bacteria and its catalyzing and synthesizing the application in D- pantoyl internal ester |
| CN114457128A (en) * | 2020-09-29 | 2022-05-10 | 安徽华恒生物科技股份有限公司 | Recombinant engineering bacterium and construction method and application thereof |
| CN112175839A (en) * | 2020-09-30 | 2021-01-05 | 遵义医科大学 | D-pantoic acid lactone hydrolase, producing strain, genetic engineering strain and application thereof |
| CN112175839B (en) * | 2020-09-30 | 2022-09-13 | 遵义医科大学 | D-pantoic acid lactone hydrolase, producing strain, genetic engineering strain and application thereof |
| CN112553226A (en) * | 2020-12-29 | 2021-03-26 | 江南大学 | Pichia pastoris gene engineering bacterium and method for preparing D-pantolactone hydrolase by using same |
| CN112553226B (en) * | 2020-12-29 | 2023-06-13 | 江南大学 | Pichia pastoris genetically engineered bacteria and method for preparing D-pantolactone hydrolase by using same |
| CN113046337A (en) * | 2021-03-18 | 2021-06-29 | 赤峰制药股份有限公司 | Pantolactone hydrolase mutant strain and application thereof |
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